Identification of phytoestrogens in protein products from an alfalfa biorefinery

Abstract

Biorefineries are complex processing facilities dedicated to the sustainable transformation of biomass into value-added products. This project studies the influence of biorefinery processing conditions on the phytoestrogen composition of protein products derived from Medicago sativa L., commonly known as alfalfa. This leguminous plant has been traditionally used as a forage and is now gaining attention for its potential in producing proteins for human consumption. Phytoestrogens are bioactive secondary metabolites present in leguminous plants. Their structural similarity to the mammalian estrogen 17β-estradiol allows them to bind to estrogen receptors in animals. Given their capacity to produce both beneficial and adverse biological effects, identifying these compounds in alfalfa-derived products is critical to ensure consumer safety. Ten alfalfa samples obtained from an industrial-scale biorefinery developed by Contento Trade Srl (Italy) were analyzed. These included nine mixed-protein and one fresh alfalfa samples, processed under varying conditions, including different harvest lots, drying delays, and drying techniques. Compound identification in the studied samples was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), supported by a custom-built database of phytoestrogens and related metabolites. Cross-referencing the obtained analytical data with the database enabled the tentative identification of 24 phytoestrogens across the samples. Comparative analysis showed that biorefinery processing conditions significantly affected the phytoestrogen profile of the mixed-protein products. Environmental stress factors affecting the fresh alfalfa material, such as morning frost exposure prior to harvest, altered these profiles. Regarding the biorefining drying conditions of the extracted mixed-proteins juice, delayed dryings of 16 or 36 hours were associated with higher phytoestrogen levels, indicating that immediate drying is preferable. In contrast, the drying technique (i.e. spray-drying or freeze-drying) had no significant effect on the final phytoestrogen composition. This finding supports the use of spray-drying as a more sustainable option for the biorefinery operation, given its lower energy and time requirements